Roger L. Clem

Calcium-Permeable AMPA Receptor Dynamics Mediate Fear Memory Erasure

Wiping Out Memories Inhibition of fear responses can be unexpectedly reversed even when a subject is perfectly safe. This can lead to inappropriate reactions to a fear-associated trigger, such as a bright light or loud noise. This type of reaction appears to underpin posttraumatic stress disorder, but there is little understanding of when training to inhibit fear may fail or succeed. Using a combination of electrophysiology and behavioral training in mice, Clem and Huganir (p. 1108, published online 28 October) observed that fear conditioning increased synaptic transmission by calcium-permeable AMPA receptors into the part of the brain that controls emotional responses (the amygdala). This effect lasted for about a week, during which the fearful memories could be erased if the animals were trained to reduce conditioned fear responses. Postmortem brain slices showed that the fear-induced synaptic changes also reversed, except in transgenic mice with a mutant subunit of the AMPA receptor. The subunit composition of AMPA receptors at lateral amygdala synapses changes after the acquisition of associative fear. Traumatic fear memories can be inhibited by behavioral therapy for humans, or by extinction training in rodent models, but are prone to recur. Under some conditions, however, these treatments generate a permanent effect on behavior, which suggests that emotional memory erasure has occurred. The neural basis for such disparate outcomes is unknown. We found that a central component of extinction-induced erasure is the synaptic removal of calcium-permeable α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs) in the lateral amygdala. A transient up-regulation of this form of plasticity, which involves phosphorylation of the glutamate receptor 1 subunit of the AMPA receptor, defines a temporal window in which fear memory can be degraded by behavioral experience. These results reveal a molecular mechanism for fear erasure and the relative instability of recent memory.

Pathway-Specific Trafficking of Native AMPARs by In Vivo Experience

An accumulating body of evidence supports the notion that trafficking of AMPA receptors (AMPARs) underlies strengthening of glutamatergic synapses and, in turn, learning and memory in the behaving animal. However, without exception, these experiments have been performed using artificial stimulation protocols, cultured neurons, or viral-overexpression systems that can significantly alter the normal function of AMPARs. Using a single-whisker experience protocol that significantly enhances neuronal responses in vivo, we have targeted neurons in and around the spared whisker column of fosGFP transgenic mice for whole-cell recording. Here we show that in vivo experience induces the pathway-specific strengthening of neocortical excitatory synapses. By assaying AMPARs for rectification and sensitivity to joro spider toxin, we find that in vivo experience induces the delivery of native GluR2-lacking receptors at spared, but not deprived, inputs. These data demonstrate that pathway-specific trafficking of GluR2-lacking AMPARs is a normal feature of synaptic strengthening that underlies experience-dependent plasticity in the behaving animal.

Secreted Semaphorins Control Spine Distribution and Morphogenesis in the Postnatal CNS

The majority of excitatory synapses in the mammalian CNS (central nervous system) are formed on dendritic spines, and spine morphology and distribution are critical for synaptic transmission, synaptic integration and plasticity. Here, we show that a secreted semaphorin, Sema3F, is a negative regulator of spine development and synaptic structure. Mice with null mutations in genes encoding Sema3F, and its holoreceptor components neuropilin-2 (Npn-2, also known as Nrp2) and plexin A3 (PlexA3, also known as Plxna3), exhibit increased dentate gyrus (DG) granule cell (GC) and cortical layer V pyramidal neuron spine number and size, and also aberrant spine distribution. Moreover, Sema3F promotes loss of spines and excitatory synapses in dissociated neurons in vitro, and in Npn-2-/- brain slices cortical layer V and DG GCs exhibit increased mEPSC (miniature excitatory postsynaptic current) frequency. In contrast, a distinct Sema3A–Npn-1/PlexA4 signalling cascade controls basal dendritic arborization in layer V cortical neurons, but does not influence spine morphogenesis or distribution. These disparate effects of secreted semaphorins are reflected in the restricted dendritic localization of Npn-2 to apical dendrites and of Npn-1 (also known as Nrp1) to all dendrites of cortical pyramidal neurons. Therefore, Sema3F signalling controls spine distribution along select dendritic processes, and distinct secreted semaphorin signalling events orchestrate CNS connectivity through the differential control of spine morphogenesis, synapse formation, and the elaboration of dendritic morphology.

Ongoing in Vivo Experience Triggers Synaptic Metaplasticity in the Neocortex

In vivo experience can occlude subsequent induction of long-term potentiation and enhance long-term depression of synaptic responses. Although a reduced capacity for synaptic strengthening may function to prevent excessive excitation, such an effect paradoxically implies that continued experience or training should not improve and may even degrade neural representations. In mice, we examined the effect of ongoing whisker stimulation on synaptic strengthening at layer 4-2/3 synapses in the barrel cortex. Although N-methyl-d-aspartate receptors were required to initiate strengthening, they subsequently suppressed further potentiation at these synapses in vitro and in vivo. Despite this transition, synaptic strengthening continued with additional sensory activity but instead required the activation of metabotropic glutamate receptors, suggesting a mechanism by which continued experience can result in increasing synaptic strength over time.

PICK1 Loss of Function Occludes Homeostatic Synaptic Scaling

Homeostatic synaptic scaling calibrates neuronal excitability by adjusting synaptic strengths during prolonged changes in synaptic activity. The molecular mechanisms that regulate the trafficking of AMPA receptors (AMPARs) during synaptic scaling are largely unknown. Here, we show that chronic activity blockade reduces PICK1 protein level on a time scale that coincides with the accumulation of surface AMPARs. PICK1 loss of function alters the subunit composition and the abundance of GluA2-containing AMPARs. Due to aberrant trafficking of these receptors, the increase in synaptic strength in response to synaptic inactivity is occluded in neurons generated from PICK1 knock-out mouse. In agreement with electrophysiological recordings, no defect of AMPAR trafficking is observed in PICK1 knock-out neurons in response to elevated neuronal activity. Overall, our data reveal an important role of PICK1 in inactivity-induced synaptic scaling by regulating the subunit composition, abundance, and trafficking of GluA2-containing AMPARs.

A seizure-induced gain-of-function in BK channels is associated with elevated firing activity in neocortical pyramidal neurons

A heritable gain-of-function in BK channel activity has been associated with spontaneous seizures in both rodents and humans. We find that chemoconvulsant-induced seizures induce a gain-of-function in BK channel current that is associated with abnormal, elevated network excitability. Action potential half-width, evoked firing rate, and spontaneous network activity in vitro were all altered 24 h following picrotoxin-induced seizures in layer 2/3 pyramidal cells in the neocortex of young mice (P13-P16). Action potential half-width and firing output could be normalized to control values by application of BK channel antagonists in vitro. Thus, both inherited and acquired BK channel gain-of-functions are linked to abnormal excitability. Because BK channel antagonists can reduce elevated firing activity in neocortical neurons, BK channels might serve as a new target for anticonvulsant therapy.

The human language-associated gene SRPX2 regulates synapse formation and vocalization in mice.

Building Vocalization The transcription factor FoxP2 (forkhead box P2) affects language acquisition in humans and regulates the protein SRPX2 (sushi repeat-containing protein X-linked 2), which itself also affects language. Sia et al. (p. 987, published online 31 October; see the Perspective by Lieberman) found that in the mouse brain, the FoxP2 transcription factor binds to the SRPX2 gene. In tissue culture experiments, FoxP2, but not SRPX2, affected dendritic morphology, while both FoxP2 and SRPX2 affected the formation of excitatory synapses. Overexpression of SRPX2 in the mouse brain equivalent of the human brains language area affected synapse density and disrupted the ultrasonic vocalizations emitted by mouse pups in search of their mothers. Studies in mice and rats elucidate the function of a protein encoded by a gene that affects language. [Also see Perspective by Lieberman] Synapse formation in the developing brain depends on the coordinated activity of synaptogenic proteins, some of which have been implicated in a number of neurodevelopmental disorders. Here, we show that the sushi repeat–containing protein X-linked 2 (SRPX2) gene encodes a protein that promotes synaptogenesis in the cerebral cortex. In humans, SRPX2 is an epilepsy- and language-associated gene that is a target of the foxhead box protein P2 (FoxP2) transcription factor. We also show that FoxP2 modulates synapse formation through regulating SRPX2 levels and that SRPX2 reduction impairs development of ultrasonic vocalization in mice. Our results suggest FoxP2 modulates the development of neural circuits through regulating synaptogenesis and that SRPX2 is a synaptogenic factor that plays a role in the pathogenesis of language disorders.

Young and old Pavlovian fear memories can be modified with extinction training during reconsolidation in humans

Extinction training during reconsolidation has been shown to persistently diminish conditioned fear responses across species. We investigated in humans if older fear memories can benefit similarly. Using a Pavlovian fear conditioning paradigm we compared standard extinction and extinction after memory reactivation 1 d or 7 d following acquisition. Participants who underwent extinction during reconsolidation showed no evidence of fear recovery, whereas fear responses returned in participants who underwent standard extinction. We observed this effect in young and old fear memories. Extending the beneficial use of reconsolidation to older fear memories in humans is promising for therapeutic applications.

PICK1 Regulates Incorporation of Calcium-Permeable AMPA Receptors during Cortical Synaptic Strengthening

While AMPA-type glutamate receptors (AMPARs) found at principal neuron excitatory synapses typically contain the GluR2 subunit, several forms of behavioral experience have been linked to the de novo synaptic insertion of calcium-permeable (CP) AMPARs, defined by their lack of GluR2. In particular, whisker experience drives synaptic potentiation as well as the incorporation of CP-AMPARs in the neocortex. Previous studies implicate PICK1 (protein interacting with C kinase-1) in activity-dependent internalization of GluR2, suggesting one potential mechanism leading to the subsequent accumulation of synaptic CP-AMPARs and increased synaptic strength. Here we test this hypothesis by using a whisker stimulation paradigm in PICK1 knock-out mice. We demonstrate that PICK1 facilitates the surface expression of CP-AMPARs and is indispensable for their experience-dependent synaptic insertion. However, the failure to incorporate CP-AMPARs in PICK1 knock-outs does not preclude sensory-induced enhancement of synaptic currents. Our results indicate that synaptic strengthening in the early postnatal cortex does not require PICK1 or the addition of GluR2-lacking AMPARs. Instead, PICK1 permits changes in AMPAR subunit composition to occur in conjunction with synaptic potentiation.

Pathway-Selective Adjustment of Prefrontal-Amygdala Transmission during Fear Encoding

Conditioned fear requires neural activity in the basolateral amygdala (BLA) and medial prefrontal cortex (mPFC), structures that are densely interconnected at the synaptic level. Previous work has suggested that anatomical subdivisions of mPFC make distinct contributions to fear expression and inhibition, and that the functional output of this processing is relayed to the BLA complex. However, it remains unknown whether synaptic plasticity in mPFC-BLA networks contributes to fear memory encoding. Here we use optogenetics and ex vivo electrophysiology to reveal the impact of fear conditioning on BLA excitatory and feedforward inhibitory circuits formed by projections from infralimbic (IL) and prelimbic (PL) cortices. In naive mice, these pathways recruit equivalent excitation and feedforward inhibition in BLA principal neurons. However, fear learning leads to a selective decrease in inhibition:excitation balance in PL circuits that is attributable to a postsynaptic increase in AMPA receptor function. These data suggest a pathway-specific mechanism for fear memory encoding by adjustment of mPFC-BLA transmission. Upon reengagement of PL by conditioned cues, these modifications may serve to amplify emotional responses.